Project Summary In nitrogenases, ironsulfur clusters transcend their usual role as electron transfer sites, by performing the multielectron reduction of N2 to NH3. This enzyme thus shows the amazing catalytic potential of ironsulfur clusters in biological systems. In addition to its unique ability to reduce N2, the FeMoco active site of nitrogenase has a carbide (C4), a feature that is new in biological chemistry. Intermediates in the biosynthesis and catalytic mechanism are likely to have hydride, carbene, N2, and hydrazine moieties, which are unknown in other enzymes. Learning the relationship between the structure and function of nitrogenase is aided by synthetic molecules that have specific similarities to the FeMoco. Though they are simplified, they make it possible to test structural features one at a time without the complication of the other cofactors and protein. Our guiding hypothesis is that carbide holds and releases lowcoordinate iron, which can form FeN2 and FeH intermediates. In this hypothesis, sulfide donors in the FeMoco give reactive highspin electronic configurations. We will test these ideas using synthetic iron clusters with combinations of sulfide, nitride, carbene and carbide bridges. Synthetic compounds with these features will show the feasibility of the proposed functional groups on ironsulfur clusters, establish the spectroscopic signatures of these functional groups, and show whether their behavior is consistent with the models for FeMoco biosynthesis and mechanism. In the proposed research, we will create synthetic ironcontaining compounds with each of the following novel functionalities: unsaturated ironsulfur clusters, ironsulfidehydride clusters, high spin ironcarbene and carbide clusters, and N2cleaving iron complexes. The isolation and characterization of these compounds is made possible by the use of bulky supporting groups. The bulky groups also facilitate crystallization, and enhance solubility in solvents that can be used at low temperature. Crystallography, kinetic studies, electrochemistry, and reactivity will be used to elucidate the atomiclevel detail of the elementary steps of smallmolecule binding and reduction. The synthetic complexes will be evaluated by ENDOR, infrared, Raman, Mssbauer, and Xray absorption spectroscopies to provide a link between the structures of novel model compounds and the known data for nitrogenases. We anticipate that the proposed work will lead to valuable precedents for reaction pathways in nitrogenases. Although much is known about the mechanisms of multielectron oxidation reactions in bioinorganic chemistry, the knowledge about multielectron biological reductions lags far behind, and there is particular need for research on smallmolecule reactions of ironsulfur clusters. Therefore, there is fundamental importance in learning how the ironsulfide cluster in nitrogenase binds and transforms small molecules that are essential for life. In the long run, understanding the mechanisms of smallmolecule reduction in biological systems may also lead to new catalysts for use in chemical synthesis, giving an even broader impact.